Non-invasive method for isolation and detection of fetal DNA

ABSTRACT

A method of detecting the presence or absence of the fetal DNA sequence of interest in fetal DNA derived from a sample of peripheral blood obtained from a pregnant woman is described. The method involves obtaining a sample peripheral blood from a pregnant woman, treating the sample of peripheral blood such that the fetal DNA present in the fetal nucleated cells is made available for detection and detecting the presence or absence of the fetal DNA sequence of interest in the available fetal DNA. The proportion of fetal nucleated cells present in the sample of peripheral blood can be increased forming a sample enriched in fetal nucleated cells prior to the detection step. The fetal DNA sequence of interest can be detected by treating the peripheral blood sample such that fetal DNA present in the sample is made available for hybridization with a DNA probe and subsequently contacting the available fetal DNA with a DNA probe hybridizable to fetal DNA of interest under hybridization conditions. The presence or absence of hybridization between the DNA probe and the fetal DNA of interest is detected as an indication of the presence or absence of the fetal DNA of interest.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.07/772,689, filed on Oct. 7, 1991, which is a continuation-in-partapplication of U.S. Ser. No. 07/706,393, filed on May 28, 1991, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/436,057,filed on Nov. 13, 1989, now abandoned, all being entitled “Non-InvasiveMethod for Isolation and Detection of Fetal DNA” by Diana W. Bianchi.The contents of all of the forementioned applications are herebyexpressly incorporated by reference.

FUNDING

Work described herein was supported by the National Institute of Healthand Children's Hospital Medical Center.

BACKGROUND

A variety of fetal cell types—platelets, trophoblasts, erythrocytes andleucocytes—cross the placenta and circulate transiently within maternalblood (Schroder, J., J. Med. Genet. 12:230-242 (1975); Douglas G. W. etal., Am. J. Obstet. Gynec., 78:960-973 (1959)). There have been numerousreports of efforts to separate fetal cells from maternal cells presentin maternal blood, but none has been successful in isolating cellssubsequently shown to contain fetal DNA. Distinguishing fetal cells frommaternal cells has not been successful for several reasons, includingthe small number of fetal cells in a maternal blood sample and the factthat morphological differences are slight (e.g., trophoblasts are theonly fetal cells which can be distinguished from maternal cells bymorphology alone).

Others report screening the peripheral blood of pregnant women for cellsof fetal origin. Fetal identification relied on the presence of a singlecytogenetic marker, the Y chromosome. Lymphocytes with a putative “XY”karyotype were found in the maternal circulation as early as 14 weeksgestation (Walknowska, J., et al., The Lancet, 1119-1122 (1979)).

The availability of flow cytometry has led many to suggest that fetalcells could be obtained through the use of a flow cytometer and thatsuch cells could be exploited for prenatal genetic diagnosis. However,although cells sorted in this manner have been said to be of fetalorigin, based on analysis of cell surface antigens, morphology, orcytogenetic criteria, there has not been confirmation that the cellscontain fetal DNA. A method by which fetal DNA could be obtained frommaternal blood during pregnancy would be valuable, particularly if itmade it possible to carry out prenatal diagnosis by a noninvasivetechnique.

DISCLOSURE OF THE INVENTION

The present invention is based, at least in part, on the discovery thatfetal nucleated cells are present in the peripheral blood of a pregnantwoman at a level which allows them to be useful in prenatal diagnosticmethods. The method of the present invention is non-invasive because aperipheral blood sample from pregnant women, not fetal blood, is used asthe source of the fetal DNA. The fetal DNA is derived from fetalnucleated cells present in the peripheral blood of a pregnant woman. Themethod of the present invention can be used to assess fetalcharacteristics (e.g. fetal sex and chromosomal abnormalities) or can beused to diagnose whether a fetus has a prenatal disease at an earlystage of the gestational period. The non-invasive method of the presentinvention does not expose the fetus or mother to risks, e.g. infection,fetal injury, and miscarriage, associated with invasive methods such asamniocentesis.

The present invention pertains to a method of detecting the presence orabsence of a fetal DNA sequence of interest in fetal DNA derived from asample of peripheral blood obtained from a pregnant woman. The methodinvolves obtaining a sample peripheral blood from a pregnant woman,treating the sample of peripheral blood such that the fetal DNA presentin the fetal nucleated cells is made available for detection anddetecting the presence or absence of the fetal DNA sequence of interestin the available fetal DNA. The proportion of fetal nucleated cellspresent in the sample of peripheral blood can be increased forming asample enriched in fetal nucleated cells prior to the detection step.The fetal DNA sequence of interest can be detected by treating theperipheral blood sample such that fetal DNA present in the sample ismade available for hybridization with a DNA probe and subsequentlycontacting the available fetal DNA with a DNA probe hybridizable tofetal DNA of interest under hybridization conditions. The presence orabsence of hybridization between the DNA probe and the fetal DNA ofinterest is detected as an indication of the presence or absence of thefetal DNA of interest.

The method of the present invention can be used to determine the sex ofa fetus by contacting the peripheral blood sample from a woman pregnantwith a fetus with a DNA probe hybridizable to fetal Y chromosomal DNA.The presence of hybridization between the DNA probe and the fetal Ychromosomal DNA can be detected as an indication of a male fetus or theabsence of hybridization can be detected as an indication of a femalefetus.

The method of the present invention also may be used for diagnosing adisease in a fetus. A sample of peripheral blood obtained from a womanpregnant with a fetus is contacted with DNA probe hybridizable to fetalDNA of interest associated with a disease under hybridizationconditions. The presence or absence of hybridization between the DNAprobe and the fetal DNA of interest is detected as an indication ofwhether the fetus has the disease.

The method of the present invention further can be used to detect achromosomal abnormality in a fetus such as a chromosomal aneuploidy,e.g., trisomy 13, trisomy 18, or trisomy 21. A sample of peripheralblood from the woman pregnant with a fetus is obtained. The fetalnucleated cells are separated from the peripheral blood sample onto asolid support forming immobilized fetal nucleated material, e.g.metaphase or interphase nuclei. The immobilized fetal nucleated materialis contacted with a DNA probe hybridizable to chromosomal fetal DNA ofinterest under hybridization conditions. The presence or absence ofhybridization between the DNA probe and the chromosomal fetal DNA ofinterest is detected as an indication of the presence or absence of achromosomal abnormality.

The method of the present invention further can be used to determinewhether a pregnancy is at risk. Fetal blood hemhorrages into thematernal blood system typically occur when a pregnancy is at riskincreasing the number of fetal cells present in the maternal blood. Aperipheral blood sample can be obtained from a pregnant woman at aselected gestational age and the number of fetal cells present in thesample can be detected. This detected number of fetal cells can becompared to a known standard representative of the number of cellspresent at the selected gestational age during a normal pregnancy. Thestandard can be established by taking peripheral blood samples from agroup of women at the selected gestational age believed to be havingnormal pregnancies.

Other aspects of this invention relate to methods of enriching theperipheral maternal blood sample and kits containing reagents used toconduct the described methods. These aspects are described in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the method of the presentinvention by which fetal nucleated cells are isolated from maternalcells and DNA within the fetal cells is assessed for the occurrence of aparticular fetal DNA sequence.

FIG. 2 is an autoradiograph of diluted male DNA amplified for 222 bpsequence. Lane 1: reagent control; lane 2: φX174 molecular weightstandard; lane 3: 100 ng; lane 4: 10 ng; lane 5: 1 ng; lane 6: 200 pcg;lane 7: 10 pcg; lane 8: 1 pcg.

FIG. 3 is a composite autoradiograph of amplified patient DNA. Lane 1:10 ng normal male; lane 2: 10 ng normal female; lane 3: reagent control;lane 4: φX174; lane 5: sorted cells from patient 1 (male fetus); lane 6:sorted cells from patient-2 (male fetus); lane 7: sorted cells frompatient 3 (female fetus); lane 8: sorted cells from patient 6 (femalefetus); lane 9: sorted cells from patient 7 (male fetus); lane 10:sorted cells from patient 8 (male fetus); lane 11: sorted cells frompatient 9 (female fetus); lane 12: cord blood from female infant whosecells were prenatally sorted in lane 8.

FIG. 4 is a diagram demonstrating the detection of Y chromosomal DNAsequences at various points of gestation in women bearing malepregnancies.

FIGS. 5A, 5B, 5C, 5D, 5E and 5F are a series of histograms obtained whenFITC-anti transferrin receptor was used to determine the presence ofmononuclear cells in samples from non-pregnant females to which malecells have been added.

FIG. 6 is a composite autoradiograph of amplified male DNA detected inTfR cells when 10²-10⁶ male cells are added to samples from non-pregnantfemales and in TfR⁻ cells when 10⁵-10⁶ male cells are added to samples,from non-pregnant females.

FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G and 7H are a series of histogramsobtained when anti HPCA-1 antibody was used to determine the presence ofmononuclear cells it samples from nonpregnant females to which malecells have been added.

FIG. 8 is a photograph illustrating a fluorescent cell due to thepositive results of in situ hybridization of the pDP97 probe for the Ychromosome to a fetal nucleated red blood cell.

FIG. 9 is a two-dimensional bivariate histogram depicting forward anglelight scatter on the X axis (an indication of cell volume) andfluorescence intensity on the Y axis (a measure of cells binding the TfRantibody. The TfR⁺ cells respresenting 1.3% of the maternal mononuclearcells are encased in a box.

FIG. 10 is a photomicrograph of a flow-sorted interphase fetal nucleusisolated from maternal blood. The single black dot representshybridization to the Y chromosome and the three white dots representhybridization to the chromosome 21s. The karotype of this nucleus is 47,XY, +21.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an in vitro method of separating orisolating fetal nucleated cells present in the blood of a pregnant woman(a maternal blood sample) from the pregnant woman's cells and ofseparating or isolating fetal DNA from maternal DNA. It further relatedto an in vitro method of prenatal detection and/or quantitation ofselected fetal DNA in fetal DNA isolated from the maternal blood sample.The method provides a noninvasive approach to detect and/or quantitatefetal DNA, such as that associated with a disease or a condition whoseassessment during gestation is desired. It also provides a noninvasivemeans by which the sex of a fetus can be determined.

In the present method, fetal nucleated cells are isolated from amaternal blood sample by means of a detectable material which binds tothe fetal nucleated cells but not to maternal cells and is thenseparated from the maternal sample, resulting in separation of the fetalnucleated cells from the sample. The fetal nucleated cells can be anyundifferentiated hematopoietic cell and, particularly, fetal nucleatederythrocytes. In one embodiment of the present method of isolation, atleast one detectably labelled monoclonal antibody specific for anantigen present on fetal nucleated cells, but not for an antigen presenton maternal cells, is combined with a maternal blood sample and, oncebound to fetal nucleated cells, is separated from the maternal sample.Alternatively, at least one detectably labelled monoclonal antibodyspecific for an antigen present on maternal cells, but not for anantigen present on fetal nucleated cells is used. In a furtherembodiment, the two types of monoclonal antibodies are used.

In the case in which the detectable label is a fluorescent molecule,separation is carried out by means of flow cytometry, in whichfluorescently-labelled molecules are separated from unlabelledmolecules. This results in separation of fetal nucleated cells, such asfetal nucleated erythrocytes, from maternal cells and, thus, of fetalDNA from maternal DNA. That this separation has occurred can be verifiedusing known techniques, such as microscopy or detection of fetalhemoglobin.

In one embodiment of the method of the present invention by which theoccurrence of a selected DNA sequence or sequences (gene(s) or geneportion(s)) in fetal DNA is determined (detected and/or quantitated),the isolated fetal nucleated cells, such as fetal nucleatederythrocytes, are treated to render DNA present in them available foramplification. Amplification of DNA from fetal nucleated cells (fetalDNA) is carried out using a known amplification technique, such as thepolymerase chain reaction (PCR). Amplified fetal nucleated cell DNA issubsequently separated on the basis of size (e.g., by gelelectrophoresis) and contacted with a selected labelled probe, such aslabelled DNA complementary to a selected DNA sequence (e.g.,complementary to an abnormal gene or gene portion, or Y-specific DNA).Detection of the labelled probe after it has hybridized to fetal DNAresults in detection of the sequence of interest in the fetal DNA.Quantitation of the hybridized labelled probe results in quantitation ofthe fetal DNA.

In a second embodiment of the present method of determining theoccurrence of a selected DNA sequence (or sequences), cells isolated asdescribed above are sorted onto a solid support, such as a slide, andscreened for chromosomal abnormalities using in situ hybridization. Inthis embodiment, a selected nucleic acid probe, such as a labelled DNAprobe for chromosomal DNA associated with a congenital abnormality, iscombined with the fetal DNA, under conditions appropriate forhybridization of complementary sequences to occur. Detection and/orquantitation of the labelled probe after hybridization results indetection and/or quantitation of the fetal DNA to which the probe hashybridized.

The following is a description of the basis for the subject method; ofthe present method of isolating nucleated fetal cells present in theblood of a pregnant woman from maternal cells and, subsequently,separating fetal DNA from maternal DNA; and of the present method ofprenatal determination of the occurrence (presence/absence orquantitation) of selected DNA in fetal cells.

Potential Sources of Fetal Genes

It has been determined that several types of fetal cells present in theblood of a pregnant woman are a source of fetal genes. In particular, ithas been shown that fetal cells, such as nucleated erythrocytes (alsoreferred to as fetal NRBC), and other undifferentiated hematopoieticprecursor cells, such as erythroblasts, lymphoblasts and myeloblasts,can be isolated or separated from maternal blood. Fetal nucleatederythrocytes were particularly selected for sorting based on thefollowing rationale:

1. In any given fetomaternal hemorrhage, no matter how small, the ratioof fetal erythrocytes to fetal lymphocytes should remain the same as inwhole fetal blood; thus, there would be 1,000 times as many red cells aswhite cells available for analysis.

2. Normal pregnant females do not usually have circulating NRBC;therefore, an isolated NRBC would a priori have a greater chance ofbeing fetal in origin.

3. The majority of pregnancies are blood group compatible, which meansthat the “transfused” NRBC would probably be tolerated by the mother andremain in her circulation.

4. Because they are nucleated, the NRBC contain a full complement offetal genes.

It has been shown that fetal nucleated erythrocytes, as well as othertypes of fetal cells, can be isolated or separated from maternal bloodand that DNA present in the isolated fetal cells can be used to assessfetal characteristics (e.g., sex, presence or absence of chromosomalabnormalities).

Advances in Molecular Biology Applied to Fetal Cell Sorting

Recent advances in molecular biology have had an enormous impact on thefeasibility of fetal cell identification. For example, fluorescent insitu hybridization can be used for this purpose.

The development of the polymerase chain reaction (PCR) (Mullis, K., atal. Cold Spring Harb. Symp. Quant. Biol., 51:263-272 (1986), with itscapacity for DNA analysis from a single cell (Li, H., et al., Nature,355:414-417 (1988); Handyside, A. H., et al., Lancet 1:347-349 (1989)),has eliminated the technical problems associated with the small numberof fetal cells in maternal blood. It makes DNA diagnosis from a singlecell possible.

As described below, fetal nucleated erythroblasts have been shown to bepresent in blood obtained from pregnant women, thus making maternalblood a useful/reliable potential source of fetal DNA; fetal nucleatedcells have been distinguished from maternal cells on the basis ofsurface antigenic characteristics, thus making it possible to Separatethe two cell types from one another; and feral DNA present in theseparated fetal nucleated cells has been analyzed and characterized.

Detection of Fetal Gene Sequences in Maternal Blood

One of the first steps in developing the present method of isolatingfetal nucleated cells from the maternal blood supply was identificationof monoclonal antibodies that permit identification and separation offetal cells from maternal cells present in blood obtained from apregnant woman. This has been done, as described in detail in theExamples. As a result, it has been determined that monoclonal antibodieswhich recognize maternal leucocytes and monoclonal antibodies whichrecognize fetal cell surface antigens are useful in separating maternaland fetal cells. The following is a brief description of monoclonalantibodies which have been shown to be useful in separating fetalnucleated cells from maternal cells present in a maternal blood sample.However, other monoclonal antibodies which distinguish between fetal andmaternal cells on the basis of surface antigenic differences, can alsobe used in the present method.

The present method requires the use of at least one type of antibodywhich is specific for (or recognizes) a surface antigen present on fetalnucleated cells, for a surface antigen present on maternal cells, butnot specific for both. That is, the present method can be carried outusing one or more antibody which distinguishes fetal nucleated cellsfrom maternal cells. The present method can be carried out using wholeblood or blood treated or processed to enrich for (increase theconcentration of) fetal nucleated cells.

Described below is the selection and successful use of monoclonalantibodies which distinguish fetal nucleated erythrocytes from maternalcells. It is to be understood, however, that in a similar manner,monoclonal antibodies which make it possible to select for another fetalnucleated cell type (or types) can be identified and used in the presentmethod to separate fetal nucleated cell types from maternal cells (and,thus, fetal DNA sources from maternal DNA).

Initial efforts focused on the elimination of contaminating maternalleucocytes in the mononuclear cell layer and identification ofmonoclonal antibodies effective in carrying out this separation, whichresults in production of a maternal sample enriched in fetal nucleatedcells.

HLe-1 (Becton-Dickinson Monoclonal center, Mountain View, Calif.,catalog #7463) is a monoclonal antibody available as a directfluorescein isothiocyanate (FITC) conjugate. It recognizes an antigenpresent on mature human leucocytes and on very immature erythrocyteprecursors, but not on mature nucleated erythrocytes (Loken, M. E., etal., Blood, 69:255-263 (1987)). Thus, maternal leucocytes are recognizedand bound, but fetal nucleated erythrocytes are not, making separationof the two possible. As described in detail in Example 1, this labelledantibody was used to eliminate maternal leucocytes in the mononuclearcell layer.

As is also described (Example 1), a combination of monoclonal antibodieshas been used for the same purpose (i.e., elimination of maternal cellsfrom the blood sample). As described, anti-monocyte antibody (M3) andanti-lymphocytes antibody (L4) have been used to remove maternal cellsfrom the mononuclear cell layer resulting from density gradientcentrifugation.

Monoclonal antibodies which recognize fetal nucleated cells but do notrecognize maternal cells were also identified. As described in detail inExample 1, a monoclonal antibody which recognizes the transferrinreceptor was identified. Erythroblasts have been shown to express thetransferrin receptor (Loken, M. R., et al. Blood, 69:255-263 (1987))antigen on their cell surfaces from the BFU-E stage until nuclearextrusion (Loken, M. R. at al., Blood, 69:255-263 (1987)). Thetransferrin receptor is also present on activated lymphocytes(Trowbridge, I. S. and M. B. Omary, Proc. Natl. Acad. Sci. USA,78:3039-3043 (1981)), certain tumor cells (Greaves, M. et al., Int. J.Immunopharmac., 3:283-300 (1981)), and trophoblast cells (Galbraith, G.M. P. et al, Blood, 55:240-242 (1980)). Thus, such an antibody isspecific for or recognizes (binds to) fetal nucleated cells, but notmaternal leucocytes. As described in Example 1, commercially availablefluorescein-conjugated monoclonal antibodies against the transferrinreceptor (TfR) were used to separate fetal nucleated erythrocytes frommaternal cells. Although the antibody is not specific for fetalnucleated erythrocytes, it facilitated their enrichment in theflow-sorted samples. Other monoclonal antibodies which are able todistinguish between fetal nucleated cells and maternal cells present ina blood sample can also be used. Such antibodies include commerciallyavailable monoclonal antibodies and those which can be produced usingknown techniques.

Separation of fetal nucleated cells from a maternal blood sample usingantibodies described above can be carried out with samples of wholeblood or a fraction of whole blood (i.e., one resulting from treatmentor processing of whole blood to increase the proportion of fetalnucleated cells present), referred to as an enriched maternal sample. Anenriched maternal sample is produced, for example, in a two-stepprocess. The maternal sample is subjected to initial separation on thebasis of size, such as by Ficoll-Hypaque density gradientcentrifugation. This results in production of a supernatant layer, whichcontains platelets; a mononuclear cell layer; and an agglutinated pelletwhich contains non-nucleated erythrocytes and granulocytes. Themononuclear layer is separated from the other layers, to produce amaternal sample which is enriched in fetal nucleated cells.

The maternal sample, whether maternal whole blood or an enrichedmaternal sample, is subjected to separation, based on surface antigenicdifferences between fetal nucleated cells and maternal cells usingantibodies described above. The maternal sample is contacted with atleast one monoclonal antibody which is specific for either fetalnucleated cells or maternal cells, but not for both and, thus, makes itpossible to separate the two types of cells. The maternal sample can becombined with a set of two or more monoclonal antibodies, each of whichis specific for either fetal or maternal cells, but not for both. Thecombination of monoclonal antibodies can be designed to enhanceseparation of the two types of cells (e.g., the combination of anti-TfRantibody and HLe-1 antibody described previously) beyond that possiblewith a single monoclonal antibody. Separation of the fetal cells iscarried out using known techniques, such as flow cytometry, use ofimmunomagnetic beads and cell panning. In general, the monoclonalantibodies have a detectable label (e.g., radioactive material,fluorophore).

In some cases, fetal nucleated cells persisting from a previouspregnancy may be present in the peripheral blood sample. Steps may betaken to eliminate or significantly reduce the number of such residualnucleated cells if found to be present. Alternatively, a type of fetalnucleated cell known to be associated with the present pregnancy may beselected for detection obviating any potential interference orcontamination of the detection method by the residual fetal nucleatedcells. For example, fetal nucleated cell type having a relatively shortlife span may be selected for detection ensuring the fetal nucleatedcell is not from a previous pregnancy. Fetal nucleated erythrocytestypically have a life span of about three months in the maternalcirculation. The above-identified steps can be conducted usingmonoclonal antibodies which recognize antigens on the respective cells.

An embodiment of the method of the present invention by which fetalcells are isolated and fetal DNA is detected is representedschematically in FIG. 1. A maternal blood sample (typically 20 ml.) isobtained, using known techniques. The sample is separated into componentlayers on the basis of size and the mononuclear cell layer, referred toas the maternal sample enriched in nucleated cells (or enriched maternalsample), is removed for further processing. The enriched maternal sampleis contacted with at least one monoclonal antibody, as described above,and the resulting fetal nucleated cell/antibody complexes are separatedusing known methods (e.g., flow cytometry, immunomagnetic beads, cellpanning). Fetal DNA is crudely extracted from the resulting complexes(e.g., by heat), thus rendering it available for hybridization withnucleic acid probes. Fetal DNA can be analyzed for a selected DNAsequence or DNA sequences, using known techniques. Prior to analysis,fetal DNA can be amplified, as needed, using known methods (e.g., PCR).

If amplification is to be carried out, the sorted samples are amplifiedfor an appropriate number of cycles of denaturation and annealing (e.g.,approximately 24-60). Control samples include a tube without added DNAto monitor for false positive amplification. With proper modification ofPCR conditions, more than one separate fetal gene can be amplifiedsimultaneously. This technique, known as “multiplex” amplification, hasbeen used with six sets of primers in the diagnosis of DMD (Chamberlain,J. S., et al., Prenat. Diagnosis, 9:349-355 (1989)). When amplificationis carried out, the resulting amplification product is a mixture whichcontains amplified fetal DNA of interest (i.e., the DNA whose occurrenceis to be detected and/or quantitated) and other DNA sequences. Theamplified fetal DNA of interest and other DNA sequences are separated,using known techniques. Subsequent analysis of amplified DNA can becarried out using known techniques, such as: digestion with restrictionendonuclease, ultraviolet light visualization of ethidium bromidestained agarose gels, DNA sequencing, or hybridization with allelespecific oligonucleotide probes (Saiki, R. K., et al, Am. J. Hum.Genet., 43 (Suppl):A35 (1988)). Such analysis will determine whetherpolymorphic differences exist between the amplified “maternal” and“fetal” samples. In one embodiment, the amplification mixture isseparated on the basis of size and the resulting size-separated fetalDNA is contacted with an appropriate selected DNA probe or probes (DNAsufficiently complementary to the fetal DNA of interest that ithybridizes to the fetal DNA of interest under the conditions used).Generally, the DNA probes are labelled (e.g., with a radioactivematerial, a fluorophore or other detectable material). After thesize-separated fetal DNA and the selected DNA probes have beenmaintained for sufficient time under appropriate conditions forhybridization of complementary DNA sequences to occur, resulting inproduction of fetal DNA/DNA probe complexes, detection of the complexesis carried out using known methods. For example, if the probe islabelled, fetal DNA/labelled DNA probe complex is detected and/orquantitated (e.g., by autoradiography, detection of the fluorescentlabel). The quantity of labelled complex (and, thus, of fetal DNA) canbe determined by comparison with a standard curve (i.e., a predeterminedrelationship between quantity of label detected and a given reading).

The present method has been used to identify Y-specific DNA in nucleatederythrocytes obtained from peripheral blood of pregnant women. This isdescribed in Example 4. Briefly, candidate fetal cells from bloodsamples obtained from 19 pregnant women were isolated by flow sorting.The DNA in these cells was amplified for a 222 base pair (bp) sequencepresent on the short arm of the Y chromosome as proof that the cellswere derived from the fetus. The amplified DNA was compared withstandardized DNA concentrations; 0.1 to 1 ng fetal DNA was obtained inthe 20 ml maternal samples. In 7/19 cases, a 222 bp band of amplifiedDNA was detected, consistent with the presence of male DNA in theisolated cells; 6/7 of these were confirmed as male pregnancies bykaryotyping amniocytes. In the case of the female fetus, DNA preparedfrom cord blood at delivery also showed the presence of the Ychromosomal sequence. In 10/12 cases where the 222 bp band was absent,the fetuses were female. Thus, the Y chromosmal sequence wassuccessfully detected in 75% of the male-bearing pregnancies,demonstrating for the first time that it is possible to isolate fetalgene sequences from maternal blood.

As described in Example 6, male (Y-specific) DNA has been detected incells sorted from pregnant women at various points in gestation.Briefly, the mononuclear cell layer was isolated from venous bloodsamples obtained from women between 11 and 16 weeks gestation.Separation was carried out using Ficoll/Hypaque density centrifugation,followed by incubation with monoclonal antibodies (Anti-TfR, anti-Leu 4and anti-Leu^(M)3) conjugated with a fluorescent marker or compound(fluorescein, phycoerythrin) and dual color analysis and flow sorting ona fluorescence-activated cell sorter. The cells that displayed greenfluorescence, but not red fluorescence (TfR positive, Leu negative, LeuM3 negative), were fetal nucleated cells and were separated from theremainder of the sample. These cells were lysed, after which the DNA wasamplified and probed for the presence of a 397 bp sequence of the Ychromosome.

The results presented in Example 6 indicate the procedure allows thedetection of the 397 bp sequence present in as little as 5 pg of maleDNA. In addition, they suggest that there is a relationship betweengestational age and detection of male DNA, as illustrated in FIG. 4.This data suggests there may be a biologic “window” for transfer offetal nucleated erythrocytes into maternal circulation.

The present method also has been used to distinguish female fetal DNAfrom maternal DNA. The two types of female DNA were distinguished usingamplification of paternal polymorphism, as described in detail inExample 7. Briefly, venous blood samples were collected from women withuncomplicated pregnancies. Separation of fetal nucleated cells wasconducted using Ficoll/Hypaque density centrifugation, followed byincubation with monoclonal antibodies (anti-TfR, anti-Leu 4 and anti-LeuM3) conjugated with a fluorescent marker (fluorescein, phycoerthyrin)and dual color analysis and flow sorting on a fluorescence-activatedcell sorter. Fetal nucleated cells identified by displaying greenfluorescence (TfR positive), but not red fluorescence (Leu-4, Leu-3negative), were collected and lysed. The DNA from the cells wasamplified and probed for paternal sequences of the highly polymorphicregion of chromosome 17, which allows the distinction of female fetalDNA from maternal DNA.

The results demonstrated that DNA sequences from the father can beidentified in the autosomal chromosomes of the fetus. Consequently, themethod of the present invention can be used to separate female fetalnucleated cells, as well as male fetal nucleated cells, from maternalblood. Thus, the method can be used for all DNA-based diagnosticprocedures currently being used in other methods, such as amniocentesis.

Further support for the present method's capability to identifyY-specific DNA in nucleated erythrocytes obtained from peripheral bloodof pregnant women is given by reconstruction experiments. As describedin Example 8, male cord blood was added to blood obtained fromnon-pregnant females to simulate the presence of fetal cells in maternalblood. Briefly, venous blood samples were collected from healthy,non-pregnant women and the mononuclear cell layers isolated byFicoll/Hypaque density centrifugation. Mononuclear cells from theumbilical cords of male infants (ranging from 10² to 10⁶ cells) wereadded to the mononuclear cell layers of the blood of non-pregnant women.The cord blood contains a large percentage of nucleated erythrocytes.The results obtained from these experiments were substantially similarto those obtained from pregnant women at various stages in gestation.Amplified sequences from the Y chromosome, consistent with the presenceof male DNA, were detected when 10² male cells were added to the femalecells.

Experiments have also indicated that fetal hematopoietic stem cells, aswell as nucleated erythrocytes, may be used in the isolation andidentification of Y-specific DNA. Example 10 describes the procedure indetail. Briefly, venous blood samples are collected from pregnant women.The mononuclear cell layer is isolated by Ficoll/Hypaque densitycentrifugation and incubated with monoclonal antibodies which areconjugated with a fluorescent marker and directed against antigens onthe surface of hematopoietic progenitor cells (see Example 11 fordiscussion of antibodies). Fluorescent cells, separated by flow sorting,have bound antibody recognizing primitive cell surface antigens, andthus, are hematopoietic precursor cells. After the cells are lysed byboiling, they are subjected to polymerase chain reaction (PCR)amplification using primers selected to amplify a 397 base pair sequencefrom the Y chromosome. This method was used to study twenty-five women,eleven of whom have confirmed male pregnancies. In eight of these elevenwomen (77%), male DNA was detected in sorted cells which showed apositive response to a human progenitor cell antigen, thus, indicatingthat the cells were undifferentiated hematopoietic stem cells. Thisexperiment confirms that fetal hematopoietic stem cells are circulatingin the mother's blood. An additional experiment with an antibody used todetect fetal lymphoblasts (CD10) indicated that in eight of 17 women,male fetal DNA was detected in CD10+ cells. Other antibodies have beenidentified which recognize human stem cell antigens, as detailed inExample 11. The data demonstrates that the types of fetal cells presentvary as the pregnancy proceeds; therefore, it may be desirable to varythe type of antibody used in cell separation, depending on the length ofgestation.

As described in Example 12, the present method has been used to analyzevenous blood from healthy pregnant women for the presence or absence ofY chromosomal DNA sequences and has been shown to be highly accurate inits ability to do so, as well as to distinguish between samples fromwomen carrying male fetuses and women carrying female fetuses. The onlypossible source of Y chromosomal DNA in maternal blood is male fetalcells (male cells carrying an X and a Y chromosome and female cellscarry two X chromosomes). As described, two antibodies were used, aloneor in combination, for this purpose: CD36, which recognizes a cellsurface antigen on nucleated erythrocytes and monocytes and glycophorinA, which recognizes an antigen present on erythrocytes. The antibodieswere conjugated, directly or indirectly, to a fluorescent dye.Fluorescent cells in the venous sample which bound one or bothantibodies were flow sorted and later amplified for Y chromosomalsequences using PCR. Of the 18 women studied, 11 had male fetuses and 7had female fetuses. Y chromosomal DNA sequences were detected in cellssorted with CD36 and/or glycophorin A antibodies in 10 of the 11 (91%)women with male fetuses and in none of the 7 women bearing females.These results demonstrate that these two antibodies are particularlyeffective in identifying fetal nucleated cells in maternal blood.

The results of the work described above and in the Examples demonstratethat nucleated fetal cells have been isolated from maternal blood;genomic DNA has been extracted from the fetal cells and identified asbeing of fetal origin; fetal genes have been amplified using PCR; andselected DNA sequences have been identified in the fetal DNA. Itdemonstrates for the first time that fetal DNA has been detected incells isolated from maternal blood.

Uses of the Present Method of Fetal Nucleated Cell Isolation and FetalDNA Characterization

Thus, it has been demonstrated that fetal DNA can be obtained from fetalnucleated cells present in a maternal blood sample. The method ofdetecting and/or quantitating fetal DNA which is represented in FIG. 1is useful as a tool for prenatal assessment (e.g., as a means forassessing chromosomal abnormalities, for determining whether DNAassociated with a disease is present, or for detecting Y-specific DNA).It is particularly useful because it is noninvasive and requires only asmall sample of blood.

Fetal DNA sequences in fetal nucleated erythrocytes, isolated asdescribed herein or by other means by which fetal nucleated cells can beseparated from a maternal blood sample, can be analyzed or assessed forthe occurrence of a DNA sequence or DNA sequences (gene(s) or geneportion(s)) which are of interest for diagnostic or other purposes. TheDNA sequence(s) or gene(s)/gene portion(s) present in fetal cells arereferred to herein as fetal DNA of interest. For example, the selectedDNA whose presence or absence is to be determined and whose quantity canalso be determined is the gene for a disease, such as cystic fibrosis,where the causative gene or gene portion has been cloned and sequenced;alternatively, it is a probe for X- or Y-specific DNA. The sameprocedure can also be used, with appropriate modifications (e.g., anappropriate DNA probe, time, temperature), to detect other genes or geneportions.

As used in a diagnostic context, such as to detect the gene known tocause cystic fibrosis, the present method is carried out as follows:Initially, a maternal blood sample (typically 20 ml.) is obtained andseparated into component layers based on relative weights (e.g., byFicoll-Hypaque density gradient centrifugation) to remove non-nucleatederythrocytes and produce a mononuclear cell layer. This results inproduction of a maternal blood sample enriched in fetal nucleatederythrocytes. The mononuclear cell layer is stained with at least oneappropriate monoclonal antibody (e.g., one which is specific for thetype of fetal nucleated cell to be separated from the sample). Forexample, a monoclonal antibody specific for fetal nucleated cells, suchas anti-TfR antibody, described above, can be used. In general, themonoclonal antibody used bears a detectable label. Alternatively, acombination of selected labelled monoclonal antibodies, such asmonoclonal antibodies specific for fetal nucleated cells (e.g., anti-TfRantibody) and monoclonal antibodies specific for maternal leucocytes(Hle-1 or L4 and M3), each labelled with a different fluorescentcompound, can be used to remove essentially all maternal cells. Labelledcells are subsequently separated from one another using a known method,such as flow cytometry. Binding of the monoclonal antibodies to cellsfor which they are specific results in production of labelled monoclonalantibody-cell complexes. For example, in the case in which anti-TfRantibodies and HLe-1 are used, fetal nucleated erythrocytes are bound byanti-TfR antibody, to produce fetal nucleated erythrocytes/anti-TfRantibody complexes, and maternal leucocytes are bound by HLe-1 antibodycomplexes. The fetal nucleated erythrocyte/anti-TfR antibody complexesare separated from maternal cell/HLe-1 antibody complexes, using, forexample, flow cytometry. The fetal cells are lysed, to produce crudelyextracted fetal DNA which is subsequently amplified, using, for example,PCR. This results in production of amplified fetal DNA, which issubsequently separated on the basis of size. Size-separated fetal DNA iscontacted with labelled DNA probes (i.e., in prenatal detection ofcystic fibrosis, a labelled DNA probe complementary to the geneassociated with cystic fibrosis). If the fetal DNA contains DNA ofinterest (in this case, the gene associated with cystic fibrosis), fetalDNA of interest/labelled probe complexes are formed.

Fetal DNA of interest/labelled probe complexes are subsequentlydetected, using a known technique, such as autoradiography. Simplepresence or absence of labelled fetal DNA of interest can be determinedor the quantity of fetal DNA of interest can be determined. In eithercase, the result is assessment of fetal DNA obtained from a maternalblood sample for selected DNA.

The occurrence of fetal DNA associated with diseases or conditions otherthan cystic fibrosis can also be detected and/or quantitated by thepresent method. In each case, an appropriate probe is used to detect thesequence of interest. For example, sequences from probes St14 (Oberle,I., et al., New Engl. J. Med., 312:682-686 (1985)), 49a (Guerin, P., etal., Nucleic Acids Res., 16:7759 (1988)), KM-19 (Gasparini, P., et al.,Prenat. Diagnosis, 9.349-355 (1989)), or the deletion-prone exons forthe Duchenne muscular dystrophy (DMD) gene (Chamberlain, J. S., et al.,Nucleic Acids Res., 16:11141-11156 (1988)) are used as probes. St14 is ahighly polymorphic sequence isolated from the long arm of the Xchromosome that has potential usefulness in distinguishing female DNAfrom maternal DNA. It maps near the gene for Factor VIII:C and, thus,may also be utilized for prenatal diagnosis of Hemophilia A. Primerscorresponding to sequences flanking the six most commonly deleted exonsin the DMD gene, which have been successfully used to diagnose DMD byPCR, can also be used (Chamberlain, J. S. et al., Nucleic Acids Res.,16:11141-11156 (1988)). Other conditions which can be diagnosed by thepresent method include β-thalassemia (Cai, S-P., et. al., Blood,73:372-374 (1989); Cai, S-P., et al., Am. J. Hum. Genet., 45:112-114(1989); Saiki, R. K., et al., New Engl. J. Med., 319:537-541 (1988)),sickle cell anemia (Saiki, R. K., et al., New Engl. J. Med., 319:537-541(1988)), phenylketonuria (DiLella, A. G., et al., Lancet, 1:497-499(1988)) and Gaucher disease (Theophilus, B., et al., Am. J. Hum. Genet.,45:212-215 (1989)). An appropriate probe (or probes) is available foruse in the present method for assessing each condition.

It is also possible to separate fetal cells from maternal cells by meansother than flow cytometry, as mentioned previously, and to analyze fetalnucleated erythrocyte DNA obtained in this way. Such separationprocedures may be used in conjunction with or independent of flowcytometry. This is advantageous because lack of access to a flowcytometer, as well as expense, could limit potential applications ofthis technique. Thus, other methods of fetal cell separation can beused. The separation method used can result in elimination of unwantedcells (“negative selection”) or isolation of rare but desirable cells(“positive selection”).

In one embodiment, the maternal cells are depleted prior to fetal cellsorting. The mononuclear cell layer is initially isolated from the bloodof pregnant women by Ficoll-Hypaque centrifugation. The resulting cellsuspension consists predominantly of maternal cells; in order to enrichthe eventual proportion of fetal cells present, the maternal cells areselectively removed by incubating the cells with antibodies attached toa solid support. Such supports include magnetic beads, plastic flasks,plastic dishes and columns. The antibodies bind antigens present on thecell surface of mature leukocytes. Thus, the majority of maternalleukocytes are eliminated by virtue of being bound to the solid support.The total number of cells remaining in the cell suspension is smaller,but the proportion of fetal cells present is larger.

Separation by immunomagnetic beads or by cell panning can also be used.In this embodiment, the mononuclear cell layer is isolated, as describedpreviously. This layer is then mixed with antibody-coated polymerparticles containing magnetic cores (e.g., “Dynabeads”). Theseimmunomagnetic beads are available coated with a variety of antibodies.For example, immunomagnetic beads coated with antibody to leucocyteantigens and antibody to mouse immunoglobulins, which can besubsequently conjugated to mouse monoclonal antibody against the humantransferrin receptor, can be used. After mixing, the rosetted cells areisolated with a magnetic particle concentrator. In one embodiment, twosets of antibody-coated immunomagnetic beads are used in succession.First, the maternal leucocytes are depleted and then the remaining TfRpositive cells are collected. Subsequent steps in the method(amplification, separation, contact with an appropriate DNA probe orprobe set) are as described for cells separated by flow cytometry.

Mueller et al. (Lancet, 336:197-200 (1990)) have described a method ofisolating placenta-derived trophoblast cells in the blood of pregnantwomen using magnetic beads. This method included mixing 1 ml ofmonoclonal antibody hybridoma culture supernatant with 2×10⁷ magneticbeads precoated with sheep antibody to mouse IgG (Fc fragment)(Dynabeads M-450, Dynal AS, Oslo, Norway), and incubated overnight atroom temperature. The coated beads were stored at 4° C. and washed threetimes in ice-cold RPMI 1640 medium containing lithium heparin (10IU/ml). The blood from the pregnant women was collected into tubescontaining 10 IU of lithium perml of whole blood, diluted 1:10 with RPMIcontaining lithium, and incubated with the antibody coated beads at 4°C. overnight. The desired cells were bound to the antibody on the bead;the beads collected by means of a cobalt-samarium magnet. Although inthis case the antibody was directed against trophoblast antigens asimilar technique can be utilized with, for example, antibody to cellsurface antigens present on fetal nucleated erythrocytes and not presenton maternal cells. An advantage to this particular technique is that aninitial step which results in mononuclear cell isolation is not added.Additionally, the magnetic beads can be used for both positive (fetalcells) and negative (maternal cells) selection.

An alternative method of isolation can be a modification of the methoddescribed by R. J. Berenson et al. (J. of Immunol. Methods, 91(1986)) inwhich the high affinity between the protein avidin and the vitaminbiotin was exploited to create an indirect immuno-adsorptive procedure.In this technique, avidin was linked to cyanogen bromide activatedsepahrose 6 MB beads and washed in an alternating fashion with couplingbuffer (0.1 M NaHCO₃ in 0.5 M NaCl at pH 8.3). and washing buffer (0.1 Msodium acetate in 0.5 M NaCl at pH 4.5) and stored at 4° C. The bloodcells were incubated with 1) murine monoclonal antibody, and 2)biotinylated goat anti-mouse immunoglobulin. A 3 ml column of gel waspacked in Pharmacia K 19/15 column. The treated cells were passedthrough the column in phosphate buffered saline containing 2% bovineserum albumin. Adherent cells were dislodged by mechanical agitation.This technique can be applied to fetal cell separation if the antibodiesused recognize fetal cell surface antigens or maternal cell surfaceantigens, but not both. Variation in methods for conjugating antibodiesto beads exist; examples include those described by Thomas andco-workers (Thomas, T. E., et al. (J. of Immuno. Methods, 120:221-131(1989)) and by deKretser and co-workers (deKretser, T. A., et al.(Tissue Antigens, 16:317-325 (1980)). The use of an antibody-boundcolumn does not require the preliminary isolation of the mononuclearcell fraction from whole blood.

Another alternative to mononuclear cell isolation is to selectively lysematernal non-nucleated erythrocytes. A number of buffers, including0.17M NH₄Cl, 0.01M Tris, pH 7.3, have been described in the literatureand are well known in isolation of hematopoietic stem cells for bonemarrow transplantation. Other buffers (“Lyse and Fix”, GenTrak) areavailable commercially.

Once the fetal cells are isolated from maternal blood, they may becultured to increase the numbers of cells available for diagnosis, ifdesired. E. Fibach et al. (Blood, 73:100-103 (1989)) have described amethod that supports the growth of human hematopoietic progenitor cells.This step-wise method involves 1) initial culture in the presence ofconditioned medium from human bladder carcinoma cells, 2) removal ofleucocytes by harvest of non-adherent cells and lysis with monoclonalantibodies and 3) reculture of cells in medium supplemented byrecombinant erythropoietin.

Other methods of separating fetal nucleated cells from maternal cellscan also be used, provided that they make it possible to differentiatebetween fetal cells and maternal cells, and to isolate one from theother.

A kit for use in carrying out the present method of isolating anddetecting fetal DNA of interest, such as a chromosomal abnormalityassociated with a disease or other condition, in a maternal blood samplecan be produced. It includes, for example, a container for holding thereagents needed; the reagents and, optionally, a solid support for usein separating fetal nucleated cell/specific antibody complexes fromother sample components or for removing maternal cells complexed with aspecific antibody. For example, reagents in a kit to be used indetecting fetal DNA of interest after amplification of fetal DNA by PCRcan include: 1) at least one antibody specific for a surface antigencharacteristic of fetal nucleated cells but not specific for a surfaceantigen characteristic of maternal leucocytes; selected DNA primers foruse in amplifying fetal DNA by PCR; and at least one DNA probecomplementary to the fetal DNA to be detected (fetal DNA of interest).The kit, as indicated, can also include a solid support to be used inseparating complexes formed from other samples components. Such solidsupport can be, for example, a glass slide, nitrocellulose filter, orimmunomagnetic beads and can have affixed thereto an antibody selectivefor the antibody present in the fetal nucleated cell/specific antibodycomplexes.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 Antibody Selection for Isolation and Sorting of FetalNucleated Erythroctes (NRBCs)

Removal of Maternal Leucocytes from Maternal Blood Using Human LeucocyteAntigen (HLe-1)

The technique of fetal NRBC isolation began with an initialFicoll-Hypaque density gradient centrifugation to remove thetremendously high number of non-nucleated erythrocytes in maternalblood. Peripheral blood was centrifuged and separated into a supernatantlayer containing platelets, a mononuclear cell layer, and anagglutinated pellet consisting of non-nucleated erythrocytes andgranulocytes. The mononuclear cell layer consisted of lymphocytes,monocytes, possible trophoblasts, and, due to their increased size anddensity, NRBCs and some reticulocytes. While the Ficoll-Hypaquecentrifugation represented an initial enrichment in the proportion offetal NRBCs present in the maternal sample, flow cytometry and cellsorting was used to improve the purity of the isolated cell population.

The mononuclear cell layer from peripheral blood samples in 63 pregnantwomen, 15 nonpregnant adults, and 39 umbilical cords, was stained withFITC-HLe-1 for flow cytometric analysis. Umbilical cord samples wereused as a substitute for whole fetal blood. Representative histogramsdisplaying fluorescence versus low-angle light scatter (an approximationof cell size) for each of the three groups were generated. Histogrampeaks were identified that corresponded to leucocytes, erythrocytes andplatelets. In 9 pregnant women, 7 nonpregnant adults and 12 umbilicalcord samples, fluorescent (HLe-1 positive) and non-fluorescent (HLe-1NEGATIVE) cell populations were sorted for detailed microscopy afterWright-Giemsh staining. While the HLe-1 positive populations were alwayscomposed of leucocytes independent of the sample source, the HLe-1negative populations differed.

In cord blood, the HLe-1 negative cells were nonnucleated and nucleatederythrocytes with occasional platelets. In the pregnant women, therewere platelets, non-nucleated erythrocytes, and a very rare NRBC. Innon-pregnant adults, only platelets and debris were seen. Thus, cordblood, with it high percentage of NRBCS, was used as a reference toestablish cell sorting parameters. Microscopy confirmed the specificityof the antibody-antigen binding and that the sorted HLe-1 negative cellswere relatively free from leucocyte contamination. These sortingparameters were utilized to isolate potential fetal NRBC on 40pregnancies.

Enrichment of Fetal NRBC in Maternal Blood Using Transferrin ReceptorAntigen (TfR)

The transferrin receptor (Newman, R., et al., Trends Biochem. Sci.1:397-399 (1982)) is a surface glycoprotein important in cellular irontransport. The TfR is present on activated lymphocytes (Trowbridge, I.S., et al., Proc. Natl. Acad. Sci. USA, 78:3039-3043 (1981)), certaintumor cells (Greaves, M. et al., Int. J. Immunopharmac., 3:283-300(1981)), and trophoblast cells (Galbraith, G. M. P., et al., Blood,55:240-242 (1980)). Erythroblasts express the TfR on their cell surfacesfrom the BFU-E stage until nuclear extrusion (Loken, M. R., et al.,Blood, 69:255-263 (1987)). Thus, TfR is an excellent “candidate antigen”for enrichment of fetal NRBCs found in maternal blood. Monoclonalantibody against TfR is available as both a fluorescein conjugate(Becton-Dickinson catalog #7513) and a phycoerythrin (PR) conjugate(gift of Dr. Michael Loken, Becton-Dickinson). The mononuclear celllayer was isolated from peripheral blood samples in 6 pregnant women, 4non-pregnant adults, and 3 newborn umbilical cords for TfR analysis andmicroscopy. Representative histograms of fluorescence versus lightscatter from these three groups were generated.

Whereas umbilical cord samples had a large population of fluorescent TfRpositive cells) that were heterogenous in size, non-pregnant adults andpregnant adults had smaller percentages of fluorescent cells thatclustered in discrete groups. In addition, there were slight differencesin the percentages of TfR positive cells in the pregnant (mean=0.83)versus non-pregnant (mean=0.32) samples studies.

Microscope studies of the TfR positive cells were performed usingWright-Giemsa stain for morphology and Kleihauer-Betke technique for thedetection of fetal hemoglobin (Kleihauer, E., et al., Klin Wochenschr.,35:637-638 (1957)). In the umbilical cord samples, large numbers ofnucleated and non-nucleated erythrocytes containing fetal hemoglobin andoccasional leucocytes were identified visually. In the pregnant women,the predominant cell types were nucleated and non-nucleated erythrocytescontaining fetal hemoglobin, although leucocytes were infrequentlyobserved. In contrast, the samples from the non-pregnant controlsconsisted almost exclusively of lymphocytes and monocytes. Becausetrophoblast cells express TfR, it was postulated that they might bepresent in the sorted population from the pregnant women; none wasdetected.

Dual Antibody Analysis

Because both antibodies enriched the proportion of NRBCs present, butdid not completely exclude other cell types in the sorted samples,combinations of antibodies were used to isolate pure populations offetal NRBCs. Preliminary dual antibody studies were performed usingPE-conjugated TfR and FITC-conjugated HLe-1. NRBCs are TfR positive andHLe-1 negative, whereas maternal leucocytes are HLe-1 positive. Theseexperiments worked well and resulted in separation of maternalleucocytes.

Thus, the work described above defined flow cytometric parameters forenrichment and sorting of NRBCs in peripheral blood from pregnant women.In addition, microscopic studies revealed that morphologic differencesoccur in mononuclear cell populations derived from venous blood samplesin pregnant versus non-pregnant adults.

EXAMPLE 2 DNA Hybridization Studies in HLe-1 Negative Cells Sorted fromMaternal Blood

To confirm fetal origin of the cells sorted as described in Example 1, Ychromosomal probes were used because it is the Y chromosome that isunquestionably fetal in origin. The assessments were designed to studywhether the presence of Y chromosomal DNA in maternal blood as detectedon autoradiographs performed antenatally correlated with the subsequentbirth of a male infant.

DNA Isolation

HLe-1 negative cells from cord blood and pregnant women were sorted intotest tubes. Conventional methods of DNA isolation as well asmodification of cruder methods (Lau, Y-F, et al. Lancet, 1:14-16 (1984);McCabe, E. R. B., et al., Hum Genet., 75:213-216 (1987) were attemptedwithout success in detecting Y chromosome derived bands on SouthernBlots. All were limited by the small numbers of cells present.

EXAMPLE 3 Direct Hybridization to Cells Deposited on Filters

In order to circumvent technical problems associated wtih DNA isolation,a method of direct DNA hybridization to cells flow sorted ontonitrocellulose filters was developed (Bianchi, D. W., et al., Cytometry,8:197-202 (1987)). In control experiments, the sex of a newborn wasdetermined from as few as 50 sorted cord blood leucocytes or 5,000 HLe-1negative cells (a mixture of nucleated and non-nucleated cells).

The methodology was then applied to detection of Y chromosomal sequencesin HLe-1 negative cells sorted from peripheral blood samples in 40 womenbetween 8½ and 38 weeks gestation. Results were the following: Dot BlotDelivered Delivered Lost To Hybridization with Y Male Female Follow-Chromosomal Probe Infant Infant up +  3  2 0 − 21 12 2

It was concluded that hybridization with this probe was not predictiveof male pregnancy. The possibility exists that there was fetal DNApresent on the filters where DNA hybridization occurred, but that thisDNA bound to the Y probe nonspecifically. Thus, the filters interpretedas “positive” for male DNA might actually have been “positive” forfetomaternal hemorrhage.

EXAMPLE 4 Use of the Polymerase Chain Reaction (PCR) to Amplify GeneSequences in Sorted Fetal Cells

PCR, which has a capacity for making 106 copies of rare target genesequences, was used to amplify gene sequences in sorted fetal cells.Optimum conditions for PCR, given the minute amounts of DNA expectedafter a fetal cell sort (approximately 1 pg to 100 ng), were determined.Experimental conditions were modified as new information becameavailable. For example, Taq polymerase was used instead of Klenowfragment of E. Coli DNA polymerase (Kogan, S. C. et al., New England J.Med. 317:990 (1987)) because of its increased specificity in DNAreplication.

Initially, studies were performed on repeated sequences from the longarm of the Y chromosome, probe Y431-Hinfa (given by Dr. Kirby Smith,Johns Hopkins University, Baltimore, Md.) and the short arm of the Ychromosome, probe Y411 (Given by Dr. Ulrich Muller, Children's Hospital,Boston, Mass.). Repeated sequences were selected because they wouldcreate a stronger amplification signal from a rare male fetal cell. Y411is identical to Y156 (Muller, U., et al., Nucleic Acids Res.,14:1325-1329 (1986)), is repeated 10-60 fold, and is absolutely Yspecific on Southern blots. Sequence Y431 has autosomal homology infemales that limited its usefulness in sex determination.

PCR Standardization

To define the minimum amount of DNA detectable in maternal blood, aseries of standardization experiments were done. DNA from male andfemale individuals was prepared in tenfold dilutions (1 pg to 1 mcg) andamplified using the standard reagents in the GeneAmpkit (Perkin-ElmerCetus cat #N801-0055) on a Perkin-Elmer DNA Thermal Cycler. Primers411-01 and 411-03 were designed to amplify a 222 base pair (bp) sequencewithin probe Y411. The number of amplification cycles varied between 18and 30. Amplified DNA samples were electrophoresed on agarose gels,transferred to nylon filters, and hybridized to ³²P-labeled Y411 probe.While it appeared possible to detect Y specific bands on autoradiographsin lanes containing as little as 10 pg of male DNA, results were oftenmuddled by the presence of amplified DNA in female lanes or controllanes containing no added DNA. The phenomenon of “false positiveamplification” has now received universal recognition (Lo, Y-M. D., etal., Lancet, 2:697 (1988); Kwok, S., et al, Nature, 339:237-238 (1989)).

Elimination of “False Positive” Amplification

Due to the limited amount of starting material in a fetal cell sort,every effort was made to eliminate background amplification in order todetermine which fetuses truly possess Y chromosomal DNA. Thus, measureswere taken to prevent aerosol contamination of male DNA. All PCRs wereperformed under sterile conditions, wearing gloves, and using positivedisplacement pipettes. All reagents were prepared in a sterile mannerand incubated overnight prior to PCR with a restriction endonucleasehaving a digestion site within the target sequence. These precautionsresulted in a significant decrease and virtual absence of false positiveamplification, as monitored by running control reactions with allreagents but no DNA.

Successful Isolation and Amplification of Fetal Gene Sequences fromNRBCs in Maternal Blood

After eliminating sources of DNA contamination and determining that aslittle as 10 pg of male DNA (1 cell 7 pg of DNA) could be detected afterPCR amplification, candidate fetal cells from the peripheral blood of 19women at 12% to 17 weeks gestation were sorted. Monoclonal antibodyagainst TfR was used to identify the presumed NRBC. The DNA in thesorted cells was amplified for the 222 bp sequence in probe Y411 asproof that the cells were derived from the fetus in male pregnancies. In7/19 cases the 222 bp band of amplified DNA was detected onautoradiographs, consistent with the presence of male DNA in theisolated cells; 6/7 of these were confirmed as male pregnancies bykaryotyping amniocytes. In the case of one female fetus, repeat studiesat 32 weeks gestation and cord blood at delivery also showed thepresence of the Y chromosomal sequence. This result might be explainedby a low level of sex chromosome mosaicism, XX/XY chimerism (Farber, C.M., et al., Hum. Genet., 82:197-198 (1989)), or the presence of the Y411sequence in single copy on the X chromosome or autosomes. In 10/12 caseswhere the 222 bp was absent, the fetuses were female. Therefore,detection of the Y chromosomal sequence was successful in 6/8 of 75% ofthe male-bearing pregnancies. In the two pregnancies where male DNA wasnot detected, there may have been fetomaternal blood groupincompatibility. Alternatively, there may not have been fetomaternalhemorrhage or the number of NRBCs present may have been below the limitof sensitivity for detection of DNA. The conditions used made itpossible to detect a minimum of 100 pg of fetal DNA, or the equivalentof 15 fetal cells. The limit of sensitivity can be improved by extendingthe number of cycles used in PCR. This work demonstrated that for thefirst time, fetal DNA was detected in cells isolated from maternalblood.

To further decrease false positive amplification and permit detection offetal DNA at the single cell level on agarose gels, PCR is being carriedout using primers derived from a single copy of sequence specific forthe long arm of the Y chromosome, PY49a (Guerin, P., et al., NucleicAcids Res., 16:7759 (1988)). In preliminary experiments using 60 cyclesof PCR, Y chromosomal DNA is visible on ethidium-bromide stained agarosegels. This extraordinary degree of sensitivity will now be applied toDNA from sorted fetal cells.

EXAMPLE 5 Determination of the Volume, Morphology and Universality ofFetomaternal Hemorrhage

a. General Strategy

It is also possible, because of the availability of the present methodof isolating fetal nucleated cells from blood obtained from a pregnantwoman, to determine whether fetal cells can be found in the maternalblood in all pregnancies. A data base can be created that can provideinformation on the number and type of fetal cells circulating inmaternal blood as pregnancy progresses. Based on previous work, it isanticipated that there will be a normal range of values that isdependent on gestational age; deviation from these values will bestudied as a potential indication of a pregnancy at risk. Specifically,large amounts of fetal blood in the maternal circulation may becorrelated with placental abnormalities, threatened miscarriage andintrauterine growth retardation.

Maternal venous blood samples are collected from pregnant women,generally prior to any invasive procedures. In general, a single 20 ml.venous blood sample will be obtained. In a subgroup of patients,permission will be sought to draw blood samples every 4 weeks to followchanges in numbers of fetal cells present. Blood is collected in EDTA,diluted 1:1 with Hanks Balanced Salt Solution (HBSS), layered over aFicoll-Hypaque column (Pharmacia) and spun at 1400 rpm for 40 minutes atroom temperature. The mononuclear cell layer will be isolated, washedtwice with HBSS and stained with fluorescent monoclonal antibodies. Forexample, this can be a combination of fluoresceinisothiocyanate-conjugated antitransferrin receptor (TfR) andphycoerythrin-conjugated anti-monocyte antibodies (M3, Becton-Dickinsoncatalog 17497) and anti-lymphocyte antibodies (L4, Becton Dickinsoncatalog #7347). The staining occurs on ice, in phosphate buffered saline(PBS) containing 2% fetal calf serum and 0.1% sodium azide. The cellsare washed in PBS prior to flow cytometry. Analysis and sorting areperformed on a Becton-Dickinson FACS-IV interfaced with a Consort 40program. Data will be acquired on the relative size and fluorescence (intwo colors) of the analyzed cells. Cells that are fluorescent in thegreen wavelength (TfR positive) and not fluorescent in the redwavelength (L4 and M3 negative) will contain the presumed fetal NRBCs.The percentage of these cells in the mononuclear cell layer are recordedand analyzed as a function of gestational age. These cells are sortedfor microscopy and PCR amplification. In addition, cells that are notfluorescent in the green wavelength (TfR negative) but are fluorescentin the red wavelength (L4 and/or M3 positive) are sorted as a presumedmaternal leucocyte population and source of maternal DNA polymorphisms.

An additional benefit of studying nucleated fetal cells in maternalblood is that the amount of fetal DNA present can be extrapolated todetermine the extent of fetomaternal hemorrhage in normal and unusualpregnancies. In the pregnancies studied, an average amount of 1 ng offetal DNA (corresponding to 150 NRBCs) was present. Using publishedvalues of the number of NRBCs per liter of fetal blood at 16 weeks,(3.6×10⁹) (Millar, D. S. et al., Prenat. Diagnosis, 5:367-373 (1985);(Forestier, F., et al., Pediatr. Res., 20:342-346 (1986)) and doingsimple algebra, these results were calculated to be consistent with 2-20μl hemorrhage of fetal blood into maternal circulation. This is atrivial amount when compared with the fetoplacental blood volume at 16weeks, about 20 ml. It is important to validate and extend these resultsto generate normative data regarding fetomaternal transfusion in earlypregnancies. It will be equally important to correlate deviations fromthe expected results with pregnancy complications.

EXAMPLE 6 Detection of Male DNA in Cells Sorted from Pregnant Women atDifferent Points in Gestation

Venous blood samples (20 ml) were collected in EDTA from healthy womenwith uncomplicated pregnancies, prior to invasive diagnostic procedures,at different points in gestation. The mononuclear cell layer wasisolated by Ficoll/Hypaque density centrifugation and incubated with themonoclonal antibodies fluorescein (FITC)-conjugated anti-TfR,phycoerythrin (PE)-conjugated anti-Leu 4 and PE-conjugated anti-Leu M3(Becton-Dickinson). Dual color analysis and flow sorting were performedon a fluorescence-activated cell sorter.

Cells that display green fluorescence but not red fluorescence (TfRpositive, Leu 4 negative, Leu M3 negative) were collected into sterilemicro test tubes and frozen at −20° C. Prior to polymerase chainreaction amplification, the cells were lysed by boiling. The polymerasechain reaction (PCR) was performed under standard conditions usingstandard reagents as described in Example 4. The primers used to amplifymaterial from the Y chromosome define a 397 base pair (bp) sequence.After PCR, the patient samples were analyzed with conventional Southernblots using ³²P labelled probe. Ethidium bromide stained agarose gelsand autoradiographs were examined for the presence of the 397 bp band,which is considered significant only if reagent controls do not revealfalse positive amplification.

Under the reaction conditions described above, it was possible to detectthe 397 bp male specific band if 5 pg of male DNA was present. This isapproximately the amount of DNA present in one cell. When excess femaleDNA (500 ng) was added to the reaction mixture, the male specific bandwas consistently detectable at 100 pg.

FIG. 4 represents a summation of samples obtained from twelve womenbearing male fetuses. These samples were taken at different times inpregnancy, and one woman was sampled twice. The data indicates thatthere is a relationship betwen gestational age and the detection of maleDNA. This implies a potential biologic “window” for the transfer offetal nucleated erythrocytes into the maternal circulation.

EXAMPLE 7 Detection of Female Fetal DNA by Amplification of PaternalPolymorphisms

Venous blood samples (20 ml) were collected in EDTA from health womenwith uncomplicated pregnancies. The mononuclear cell layer was isolatedby Ficoll/Hypaque density centrifugation and incubated with themonoclonal antibodies fluorescein (FITC)-conjugated anti-TfR,phycoerythrin (PR)-conjugated anti-Leu 4 and PE-conjugated anti-Leu M3(Becton-Dickinson). Dual color analysis and flow sorting were performedon a fluorescence-activated cell sorter.

Cells that display green fluorescence but not red fluorescence (TfRpositive, Leu 4 negative, Leu M3 negative) were collected into sterilemicro test tubes and frozen at −20° C. Additionally, cells thatdisplayed red fluorescence but not green fluorescence (TfR negative, Leu4 positive, Leu M3 positive) were collected in an identical manner.Prior to polymerase chain reaction (PCR) amplification, the cells werelysed by boiling. PCR was performed using buffers containing 1 mMMgC^(l)2. The primers used in PCR amplify a highly polymorphic region ofchromosome 17. Amplified DNA sequences correspond to blocks of genestransmitted directly from parent to child. As a result of the highdegree of individual variation in these sequences, it is uncommon fortwo parents to manifest identical DNA patterns. Thus, it is possible todemonstrate inheritance of the paternal sequences in the sorted fetalcells. Since these sequences are from chromosome 17, they areindependent of fetal sex, and may be used to distinguish female fetalDNA from maternal DNA. Amplified DNA was separated by electrophoresisthrough ethidium bromide stained agarose gels. The DNA was transferredto nylon filters and probed using ³²P labeled sequence. The maternalDNA, paternal DNA TfR⁺ cells, and TfR⁻ cells were then compared.

In 5 of 10 pregnant women, it was possible to show the presence ofpaternal sequences in the sorted candidate fetal cell population. In theother 5 women, no differences were seen between the maternal DNA and theDNA obtained from the candidate fetal cells.

EXAMPLE 8 Reconstruction Experiments Using Non-Pregnant Female Blood andAdded Male Cord Blood to Simulate the Presence of Fetal Cells inMaternal Blood

Venous blood samples (20 ml) were collected in EDTA from healthynon-pregnant women. Umbilical cord blood samples (10 ml) were collectedin EDTA from normal newborns. The mononuclear cell layer was isolated byFicoll/Hypaque density centrifugation. Cell counts were performed with ahemocytometer. Separate aliquots of cells were made containing: 1)female cells alone; 2) female cells plus 10² added male cord bloodcells; 3) 3 female cells plus 10³ added male cord blood cells; 4) femalecells plus 10⁴ added male cord blood cells; 5) female cells plus 10⁵added male cord blood cells; 6) female cells plus 10⁶ added male cordblood cells; 7) male cord blood cells alone. The separate aliquots werethen incubated with the individual monoclonal antibodies being tested.Analysis and sorting were performed using a flow cytometer. For eachaliquot, a bivariate histogram was obtained, and gating parameters wereestablished for antibody positive and antibody negative cells. Thesorted cells were collected into sterile micro test tubes and frozen at−20° C. PCR amplification was performed with primers that detect a 397bp sequence unique to the Y chromosome. The presence of a band at 397 bpin autoradiographs was used to confirm the presence of male umbilicalcord blood cells in sorted samples.

FIG. 5 shows the histograms obtained when FITC-anti transferrin receptoris used. In the non-pregnant female, 0.1% of the mononuclear cells reactwith the antibody. In male cord blood, 24.9% of the mononuclear cellsreact with the antibody. With the addition of more and more umbilicalcord cells to the non-pregnant female cells, and increased percentage ofcells that react with the antibody is seen.

FIG. 6 shows that male DNA is detected in the TfR⁺ cells when 10²-10⁶male cells are added. Male DNA is detected in the TfR⁻ cells when10⁵-10⁶ male cells are added. This results from the presence of malewhite blood cells in the TfR⁻ population.

FIG. 7 shows the histograms obtained when anti HPCA-1 antibody is used.In the non-pregnant female, 0.9% of the mononuclear cells react withantibody. In umbilical cord blood, a well-defined population of cells isseen, but the percentage is only 1.1%. Thus, the addition of umbilicalcord blood cells to the nonpregnant female cells is not seen on thehistograms as clearly as with the transferrin receptor antibody. Anincreased number of HPCA⁺ cells were collected as the amounts of addedcord blood cells increased.

In agarose gels, the 397 bp band consistent with DNA was detected in theHPCA⁺ cells when 10³-10⁵ male cells were added to the female cells. MaleDNA was detected in agarose gels in the HPCA cells when 10⁶ male cellswere added to the female cells.

EXAMPLE 9 In Situ Hybridization Using Molecular Probes RecognizingIndividual Chromosomes in Flow Sorted Nucleated Erythrocytes

To demonstrate diagnostic utility of the present invention, a DNA probeset was constructed of chromosome specific probes that provided bothgood signal to noise ratios and good spatial resolution of thefluorescent signals. Accordingly, specific probes were developed forfive chromosomes frequently seen as liveborn aneuploidies; chromosomes13, 18, 21, X and Y. A probe for chromosome 1 was used as a control. Inconstructing the probes, the general strategy was to identify a startingclone that mapped to the desired chromosomal region by multiple geneticand physical methods, and then to use that clone to identify a matchingcosmid “contig” which was then used as a hybridization probe.

Hybridization of the high copy number repeat sequences was suppressed byinclusion of total genomic human DNA, and the chromosomal specificityverified by hybridization to metaphase spreads. The probes gave sharp,punctate fluorescent signals in interphase cells that were easilydiscriminated and enumerated. The Y probe used in this study was pDP97,a repetitive clone (a 5.3 kb EcoRI Y fragment from cosmid Y97 subclonedinto EcoRI site of pUC-13). All probes were labeled with biotin,hybridized under suppression conditions, and specific hybridizationdetected by conjugated strepto-avidin-FITC, which showed as a single“dot” in the FITC image. As illustrated in FIG. 8, the Y chromosome wasdetected by in situ hybridization of a pDP97 probe for the Y chromosomein a fetal nucleated red blood cell. Thus, prenatal diagnosis forchromosomal abnormalities could be performed on fetal cells isolatedfrom maternal blood.

EXAMPLE 10 Detection of Fetal Hematopoietic Stem Cells in MaternalCirculation

Venous blood samples (20 ml) were collected in EDTA or citrate dextrosefrom healthy women with uncomplicated pregnancies, prior to invasivediagnostic procedures, at different points in gestation. The mononuclearcell layer was isolated by Ficoll/Hypaque density centrifugation andincubated with monoclonal antibodies directed against antigens expressedon the cell surface of hematopoietic progenitor cell. These antibodiesare described in Example 2. The antibodies were either directly orindirectly conjugated to a fluorescent dye. Analysis and flow sorting offluorescent cells were performed on a fluorescence-activated cellsorter. Fluroescent cells are cells that have bound antibody recognizingprimitive cell surface antigens; thus, they are hematopoietic precursorcells. These cells were physically sorted into sterile micro test tubesand frozen at −20° C. Prior to polymerase chain reaction amplification,the cells were lysed by boiling. The polymerase chain reaction (PCR) wasperformed under standard conditions using standard reagents. The primersselected amplified material from the Y chromosome as a means ofdetecting male fetal cells. These primers defined a 397 base pairsequence. After PCR, the patient samples were analyzed with conventionalSouthern blots using a ³²P labeled probe. Ethidium bromide stainedagarose gels and autoradiographs were examined for the presence of theamplified 397 bp band, which was considered significant only if reagentcontrols did not reveal false positive amplification.

Twenty-five women were studied with antibody to a human progenitor cellantigen (HPA). This antigen has been given a cluster of differentiation(CD) designation as CD34. Cells that are CD34+ are undifferentiated andrepresent hematopoietic stem cells. Eleven of the twenty five womenwhose peripheral blood was sorted have confirmed male pregnancies. In8/11 (72%) of those women, male DNA was detected in the sorted CD34+cells. This confirms that fetal hematopoietic stem cells are circulatingin mother's blood.

Additionally, antibodies were used against the oncofetal antigenexpressed in many leukemias (CALLA) to detect fetal lymphoblasts in thematernal circulation. This antibody is designated as CD10. Seventeenwomen have been sorted with antibody to CD10. In 8/17, evidence of malefetal DNA has been detected in CD10+ cells.

EXAMPLE 11 Additional Antibodies to Detect Fetal Hematopoietic Cells inMaternal Circulation

Human fetal umbilical cord blood is being used as a source ofmononuclear cells for study of fetal cell populations, in order todetermine additional antibodies to detect fetal hematopoietic cells inmaterial circulation. Pure fetal blood samples were tested from as earlyas 19 weeks gestation.

The mononuclear cell layer is isolated as described in example 10.Mononuclear cells are incubated with mouse monoclonal antibodies tohuman stem cell antigens (CD34, CD10, CD38, which recognizesmyeloblasts, CD33, HLA-DR, CD36, glycophorin A, CD71, 8G12, THB-7 andothers). Many of these antibodies are available directly conjugated to afluorescent dye. If the antibody is not fluorescent, a sandwichtechnique is used to attach fluorescein-conjugated goat anti mouseantibody to the primary antibody.

Our data thus far has demonstrated that there are differences in thetypes of fetal cells present as the pregnancy proceeds. Approximately 4%of the fetal mononuclear cells are CD10+ until 34 weeks gestation. CD10+cells are not detectable at term. Approximately 5% of fetal mononuclearcells are CD34+. Between 8 and 18% of fetal mononuclear cells are CD38+.The percentages of CD38+ cells increase during the pregnancy. Therefore,varying the type of antibody used in cell separation based on the lengthof gestation may help increase the isolation of fetal cells.

EXAMPLE 12 Detection of Y Chromosomal DNA Sequences in Venous BloodSamples Obtainly Only from Women Having Male Fetuses

Venous blood samples (20 ml) were collected in EDTA or citrate dextrosefrom healthy women with uncomplicated pregnancies, prior to invasivediagnostic procedures, at different points in gestation. The mononuclearcell layer was isolated as described in Example 10. Alternatively,mononuclear cells cobld be obtained by (nonnucleated) red cell lysisbuffers. In this example, the specific monoclonal antibodies chosen tolabel the fetal cells of interest are CD36 and glycophorin A, usedsingly or in combination. These antibodies are either directly orindirectly conjugated to a fluorescent dye. CD36 recognizes a cellsurface antigen present on nucleated erythrocytes and monocytes, whereasglycophorin A recognizes an antigen present on erythrocytes. Fluorescentcells that have bound either or both antibodies were flow sorted; suchcells were frozen and subsequently amplified for Y chromosomal sequencesby the polymerase chain reaction as described in Example 10.

In a group of 18 women studies, 11 had male fetuses and 7 had femalefetuses. Y chromosomal DNA sequences were detected in the cells sortedwith CD36 and/or glycophorin A antibodies in 10/11 (91%) of the womenhaving males. None of the women bearing females (0/7) had Y chromosomalDNA sequences detected. The probability of obtaining these results bychance, p=0.00025 by Fisher exact test. Thus, the antibodies CD36 andglycophorin A are particularly effective in identifying fetal nucleatedcells circulating in maternal blood.

EXAMPLE 13 Detection of Fetal Cells with 47 XY +21 Karotype in MaternalPeripheral Blood

A peripheral blood sample (20 ml.) was obtained from a pregnant woman atnineteen weeks of gestation. A previous ultrasound examination performedat 17 weeks gestation revealed fetal malformations consistent with adiagnosis of Down Syndrome. An amniocentesis was performed for fetalchromosome analysis and results of the karotype were 47, XY, +21.

Flow Cytometry

The woman's venous blood was collected in ethylenediamine tetraaceticacid (EDTA), diluted 1:3 with Hanks' balanced salt solution, layeredover a Ficoll/Hypaque column (Pharmacia) and spun at 2000 rpm for twentyminutes at room temperature. The mononuclear cell layer was isolated,washed with phosphate buffered saline (PBS) and centrifuged at 1400 rpmfor ten minutes at 4° C. The cell pellet was incubated with a 1:10dilution of fluorescein-conjugated anti-TfR (anti CD 71)Becton-Dickinson Catalog No. 7513) in PBS on ice for thirty minutes. Thecells were washed once in PBS prior to flow sorting.

Analysis and sorting were performed on a Becton-Dickinson FACS IV with aConsort 40 program as described (Bianchi et al., Cytometry 8:197-202(1987)). The gain was standardized manually using fluorescent beads anda fluorescein isothiocyanate (FITC) conjugated antibody control, keyholelimpet hemocyanin, an antigen not expressed on human cells(Becton-Dickinson Catalog No. 9041). A small aliquot of the woman'smononuclear cells were incubated with the antibody control to determinebackground fluorescence. TfR⁺ (fluorescent) and TfR⁻ (non-fluorescent)cells were determined by physical separation on a logarithmic scale andsorted into 1.5 ml centrifuge tubes.

Fluorescence In Situ Hybridization

A solution of methanol and acetic acid (3:1) was used to fix nuclei fromsorted cells to glass slides which were then stored at −20° C. (Klingeret al., Am. J. Hum. Genet. 51:55-65 (1992)). The contents of the entireforementioned Klinger et al. reference is expressly incorporated byreference. Prior to hybridization, slides were warmed briefly at 60° C.Control hybridizations with male lymphocytes were performedconcurrently.

The probe sets used for hybridization to the Y chromosome and tochromosome 21 have been described previously (Klinger et al., citedsupra, (1992)). The Y chromosome probe was labeled with biotin-dUTP(Sigma) and the chromosome 21 probe was labeled with digoxigenin-dUTP(Boehringer Mannheim). The probes were hybridized simultaneously undersuppression conditions (Cremer et al., Hum Genet 80:235-246 (1988);Lichter et al., Hum Genet 80:224-234 (1988)). The Y chromosome probe wasdetected with avidin conjugated to Texas Red (Vector Laboratories) andthe chromosome 21 probe was detected with anti-digoxigenin conjugated tofluorescein isothiocyanate (FITC) (Boehringer Mannheim). The slides weremounted in 2.33% DABCO (1,4-diazobicyclo[2.2.2]octane) (Sigma) in 100 mMTris-HCl, pH 8.0, 90% (v/v) glycerol with 0.5 ug/ml4,6-diamidino-2-phenyl-indole (DAPI) as a counterstain (Sigma). Slideswere analyzed using a Zeiss Axioplan epifluorescence microscope. FITCand Texas Red were monitored simultaneously using a dual band passfilter set (Omega Optical, Inc., Brattleboro, Vt.). Images were capturedwith a cooled confocal digital camera (Photometrics Ltd., Tucson, Ariz.)and image processing was performed with software developed byRecognition Technology, Inc., (Westboro, Mass.).

The results of the contour plot depicting fluorescence intensity versuslight scatter are shown in FIG. 9. The percentage of TfR⁺ cells was 1.3.A total of approximately 43,000 TfR⁺ cells and 300,000 TfR⁻ cells weresorted.

In situ hybridization studies performed on the TfR⁻ cells revealed thepresence of numerous nuclei. Control slides prepared from malelymphocytes gave bright signals with both the 21 and Y probes. Thenuclei from TfR⁻ cells that hybridized to the probes were exclusivelymaternal, with two signals for chromosome 21 and no signal with the Y.The majority of the sorted cells in the TfR⁺ fraction did not hybridize,due to the fact that most of them were reticulocytes, which areanucleate. Most of the nuclei present were in a few giant clumps thathad retained large amounts of cytoplasm. Of the few TfR⁺ cells that didhybridize, FITC and Texas Red signals were generally weak. There werethree nuclei with one Texas Red and three FITC signals, consistent withthe presence of one Y chromosome and three copies of chromosome 21 (FIG.10). The remainder of the data are summarized as follows.

Results of hybridization studies on scored cells: Texax Red (Y) FITC(21) Number of Cells Number 1 3 3 of signals 1 0 8 0 3 1 1 1 1 13Uninformative 15 28Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of detecting the presence or absence of a fetal DNA sequenceof interest in fetal DNA derived from a sample of peripheral bloodobtained from a pregnant woman, comprising: obtaining a sample ofperipheral blood from a pregnant woman; treating the sample ofperipheral blood such that fetal DNA present in fetal nucleated cellspresent in the sample is made available for detection resulting inavailable fetal DNA; and detecting the presence or absence of a fetalDNA sequence of interest in the available fetal DNA.
 2. The method asclaimed in claim 1 wherein the proportion of fetal nucleated cellspresent in the sample of peripheral blood obtained from the pregnantwoman is increased forming a sample enriched in fetal nucleated cellsprior to the detection step.
 3. The method of claim 1 wherein the sampleenriched in fetal nucleated cells is formed by separating thenon-nucleated cells from the nucleated in the peripheral blood sampleforming a nucleated cell enriched sample and by treating the nucleatedcell enriched sample such that the proportion of fetal cells in thesample is increased forming a fetal nucleated cell enriched sample. 4.The method of claim 1 wherein the fetal DNA is amplified prior to thedetection step resulting in amplified fetal DNA.
 5. A method ofdetecting the presence or absence of fetal DNA of interest in fetal DNAderived from a sample of peripheral blood obtained from a pregnantwoman, comprising: obtaining a sample of peripheral blood from thewoman; treating the sample of peripheral blood such that fetal DNApresent in fetal nucleated cells present in the sample is made availablefor hybridization with a DNA probe resulting in available fetal DNA;contacting the available fetal DNA with a DNA probe hybridizable tofetal DNA of interest under hybridization conditions; and detecting thepresence or absence of hybridization between the DNA probe and the fetalDNA of interest as an indication of the presence or absence of the fetalDNA of interest.
 6. The method of claim 5 wherein the fetal nucleatedcells in the treating step are undifferentiated hematopoietic cells. 7.The method of claim 5 wherein the fetal nucleated cells are selectedfrom the group consisting of erythroblasts, lymphoblasts, andmyeloblasts.
 8. The method of claim 6 wherein the undifferentiatedhematopoietic cells are erythroid cells.
 9. The method of claim 8wherein the undifferentiated erythroid cells are fetal nucleatederythrocytes.
 10. The method of claim 5 wherein the proportion of fetalnucleated cells present in the sample of peripheral blood obtained fromthe pregnant woman is increased forming a sample enriched in fetalnucleated cells prior to the detection step.
 11. The method of claim 10wherein the sample enriched in fetal nucleated cells is formed byseparating non-nucleated cells from nucleated cells in the peripheralblood sample forming a nucleated cell enriched sample and by treatingthe nucleated cell enriched sample such that the proportion of fetalcells in the sample is increased forming a fetal nucleated cell enrichedsample.
 12. The method of claim 5 wherein the fetal DNA of interest inthe contacting and detecting steps is Y chromosomal DNA.
 13. The methodof claim 5 wherein the hybridization in the detecting step is betweenthe DNA probe and a disease causing mutation.
 14. The method of claim 13wherein the disease causing mutation is a cystic fibrosis-causingmutation.
 15. The method of claim 13 wherein the disease causingmutation is a Duchenne muscular dystrophy-causing mutation.
 16. Themethod of claim 13 wherein the disease causing mutation is a hemophiliaA-causing mutation.
 17. The method of claim 13 wherein the diseasecausing mutation is a Gaucher disease-causing mutation.
 18. The methodof claim 13 wherein the disease causing mutation is a sickle cellanemia-causing mutation.
 19. The method of claim 13 wherein thehybridization in the detecting step is between the DNA probe and a fetalDNA of interest selected for assessing a chromosomal abnormality. 20.The method of claim 5 wherein the hybridization in the detecting step isin situ hybridization.
 21. The method of claim 6 wherein thehybridization in the detecting step is in situ hybridization.
 22. Themethod of claim 9 wherein the hybridization in the detecting step is insitu hybridization.
 23. The method of claim 12 wherein the hybridizationin the contacting step is in situ hybridization.
 24. The method of claim5 wherein the fetal DNA is amplified prior to the detection stepresulting in amplified fetal DNA.
 25. The method of claim 6 wherein thefetal DNA is amplified prior to the detection step resulting inamplified fetal DNA.
 26. The method of claim 9 wherein the fetal DNA isamplified prior to the detection step resulting in amplified fetal DNA.27. The method of claim 3 further comprising determining the gestationalage at the time the sample of peripheral blood is obtained and treatingthe sample such that fetal DNA present in a selected type of fetalnucleated cells is made available for hybridization, said type of fetalnucleated cell being selected based upon the known presence of suchfetal nucleated cells in the peripheral blood of a pregnant woman at thedetermined gestational age.
 28. The method of claim 5 further comprisingthe step of treating the peripheral blood sample to remove residualfetal nucleated cells from a previous pregnancy prior to the detectionstep.
 29. The method of claim 5 wherein the peripheral blood sample inthe obtaining step is obtained between about ten and about nineteenweeks gestation.
 30. A method of detecting a fetal DNA sequence ofinterest in fetal DNA derived from a sample of peripheral blood obtainedfrom a pregnant woman, comprising: obtaining a sample of peripheralblood from a pregnant woman; separating fetal nucleated cells from theperipheral blood onto a solid support forming immobilized fetalnucleated material; contacting the immobilized fetal nucleated materialwith a DNA probe hybridizable to fetal DNA of interest underhybridization conditions; and detecting the presence of hybridizationbetween the DNA probe and the fetal DNA of interest as an indication ofthe presence or absence of the fetal DNA of interest.
 31. The method ofclaim 30 wherein the hybridization in the detecting step is in situhybridization.
 32. The method of claim 31 wherein the hybridization ofhigh copy number repeat sequences present in the fetal DNA is suppressedin the contacting step.
 33. A method for determining the sex of a fetus,comprising: obtaining a sample of peripheral blood from a woman pregnantwith a fetus; treating the sample of peripheral blood such that fetalDNA present in fetal nucleated cells present in the sample is madeavailable for hybridization resulting in available fetal DNA; contactingthe available fetal DNA with a DNA probe hybridizable to fetal Ychromosomal DNA under hybridization conditions; and detecting thepresence of hybridization between the DNA probe and the fetal Ychromosomal DNA as an indication of a male fetua or the absence ofhybridization as an indication of a female fetus.
 34. A method fordiagnosing a disease in a fetus, comprising: obtaining a sample ofperipheral blood from a woman pregnant with a fetus; treating the sampleof peripheral blood such that fetal DNA present in fetal nucleated cellspresent in the peripheral sample is made available for hybridizationresulting in available fetal DNA; contacting the available DNA with aDNA probe hybridizable to fetal DNA of interest associated with adisease under hybridization conditions; and detecting the presence orabsence of hybridization between the DNA probe and the fetal DNA ofinterest associated with the disease as an indication that the fetus hasthe disease.
 35. A method for detecting a chromosomal abnormality in afetus, comprising: obtaining a sample of peripheral blood from a womanpregnant with a fetus; separating fetal nucleated cells from theperipheral blood sample onto a solid support forming immobilized fetalnucleated material; contacting the immobilized fetal nucleated materailwith a DNA probe hybridizable to chromosomal fetal DNA of interest underhybridization conditions; and detecting the presence or absence ofhybridization between the DNA probe and the chromosomal fetal DNA ofinterest as an indication of the presence or absence of a chromosomalabnormality.
 36. The method of claim 35 wherein the hybridization in thedetecting step is in situ hybridization.
 37. The method of claim 36wherein the chromosomal abnormality is a chromosomal aneuploidy.
 38. Themethod of claim 37 wherein the chromosomal aneuploidy is trisomy
 21. 39.The method of claim 37 wherein the chromosomal aneuploidy is trisomy 18.40. The method of claim 37 wherein the chromosomal aneuploidy is trisomy13.
 41. A method for determining whether a pregnancy is at risk,comprising: obtaining a peripheral blood sample from a pregnant woman ata selected gestational age; detecting the number of fetal cells presentin the peripheral blood sample; comparing the number of fetal cellsdetected to a standard to determine whether the number of fetal cells isindicative of a pregnancy at risk, said standard being selected based onthe number of fetal cells present in a peripheral blood sample obtainedfrom a woman having a normal pregnancy at the selected gestational age.42. A method of increasing the proportion of fetal nucleated cellspresent in a peripheral blood sample containing nucleated andnon-nucleated cells, comprising: separating non-nucleated cells fromnucleated cells present in a peripheral blood sample forming a nucleatedcell enriched sample; and treating the nucleated cell enriched samplesuch that the proportion of fetal cells in the sample is increasedforming a fetal nucleated cell enriched sample.
 43. The method of claim42 wherein the separating step is a density gradient centrifugation. 44.The method of claim 42 wherein the nucleated enriched sample is treatedby contacting the sample with (i) a first monoclonal antibody whichrecognizes fetal nucleated cells but not maternal cells and/or (ii) asecond monoclonal antibody which recognizes maternal cells but not fetalnucleated cells, under conditions appropriate for antibody bindingthereby producing (i) fetal nucleated cell-first monoclonal antibodycomplexes and/or (ii) maternal cell-second monoclonal antibodycomplexes, respectively, and (i) separating fetal nucleated cell-firstmonoclonal antibody complexes from maternal cells and/or (ii) separatingmaternal cell-second monoclonal antibody complexes from fetal nucleatedcells thereby separating fetal nucleated cells from maternal cells. 45.The method of claim 43 wherein the nucleated enriched sample is treatedby contacting the sample with (i) a first monoclonal antibody whichrecognizes fetal nucleated cells but not maternal cells and/or (ii) asecond monoclonal antibody which recognizes maternal cells but not fetalnucleated cells, under conditions appropriate for antibody bindingproducing (i) fetal nulceated cell-first monoclonal antibody complexesand/or (ii) maternal cell-second monoclonal antibody complexes,respectively and, (i) separating fetal nucleated cell-first monoclonalantibody complexes from maternal cells and/or (ii) separating maternalcell-second monoclonal antibody complexes from fetal nucleated cellsthereby separating fetal nucleated cells from maternal cells.